The invention relates generally to fluorescent proteins and more particularly to compositions and methods for measuring the response of a sensor polypeptide to an environmental (e.g., biological, chemical, electrical or physiological) parameter.
Fluorescent Ca2+ indicators such as fura-2, indo-1, fluo-3, and Calcium-Green have been the mainstay of intracellular Ca2+ measurement and imaging (see, for example, U.S. Pat. Nos. 4,603,209 and 5,049,673). These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentation in organelles, and perturbation of the indicators by cellular constituents. Although the Ca2+-indicating photoprotein aequorin is targetable, the photoresponse to Ca2+ is low since it is chemiluminescent. Moreover, aequorins need to incorporate exogenous coelenterazine.
Many effects of Ca2+ in cells are mediated by Ca2+ binding to calmodulin (CaM, which causes CaM to bind and activate target proteins or peptide sequences. Based on the NMR structure of CaM bound to the 26-residue M13 Ca2+-binding peptide of myosin light-chain kinase, Porumb et al. fused the C-terminus of CaM via a Gly-Gly spacer to M13. Ca2+ binding switches the resulting hybrid protein (CaM-M13) from a dumbbell-like extended form to a compact globular form similar to the CaM-M13 intermolecular complex (see, Porumb, T., et al., Prot.Engineering 7:109-115 (1994)).
Measurement of a binding member concentration in vitro or in vivo by non-invasive techniques can help elucidate the physiological function of the binding member. This can also aid in identifying changes that occur in a cell or organism in response to physiological stimuli. For example, cyclic AMP can be detected by fluorescence resonance energy transfer between separately labeled proteins that associate with each other but are not covalently attached to each other. See, U.S. Pat. No. 5,439,797.
The Aequorea victoria Green Fluorescent Protein (GFP) is useful as a marker for gene expression, as a fluorescent tag to aid in visualizing protein trafficking, and as a component of indicator systems that allow fluorescent sensing of small molecules and pH. Currently, GFPs use as a biosensor is limited to those systems that use GFP fusion proteins as partners for fluorescence resonance energy transfer (FRET) or those that use the subcellular redistribution of GFP fusion proteins as indicators of substrate concentration or the measurement of pH.
Currently, fluorescent molecules designed to measure interactions of proteins rely on cameleon molecules of tandem GFP constructs. In these constructs, conformational changes occur and alter the FRET between the GFPs and a ratiometric color change is noted. Such cameleon or FRET-sensitive constructs are large molecules in which protein conformation influences FRET efficiency of two GFPs of different colors. Although insertions into Green Fluorescent Protein have been attempted (see Abedi et al., Nucleic Acids Research, 26(2):623-630 (1998)) such insertions have been made to optimize the presentation of short peptide libraries and not to present binding molecules or sensor polypeptides. Additionally, such insertions have been only short insertions of about six amino acids in length. Until now, however, it has not been possible to make a single GFP molecules"" fluorescence sensitive to a substrate other than hydrogen ions. There currently is a desire for smaller constructs useful in measuring interactions of molecules in vitro and in vivo.
The inventors have discovered that when a sensor polypeptide is inserted into an Aequorea-related fluorescent protein (e.g., Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP) or Cyan Fluorescent Protein (CFP)) to form a construct, interaction of the sensor polypeptide with a biological, chemical, electrical or physiological parameter, for example, results in a change in fluorescence of the fluorescent protein. Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in vivo or in measuring particular characteristics of a cell (e.g., redox potential, intracellular ion concentration). These constructs rely on the responsiveness of a sensor polypeptide inserted within a GFP-sensor-related protein itself to influence the actual fluorescence of the fluorophore and not the interaction of tandem fluorescent molecules.
Accordingly, the present invention provides an isolated nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide. The fluorescent protein moiety can be any fluorescent protein, for example, an Aequorea-related fluorescent protein moiety. More specifically, the Aequorea-related fluorescent protein can be, for example, a GFP, CFP or YFP moiety. The sensor polypeptide may be any polypeptide moiety, for example, a moiety that undergoes a conformational change upon interaction with a molecule, oxidation-reduction, or changes in electrical or chemical potential. The indicator may further include a linker moiety, linking the N- and C-terminal amino acids of the sensor polypeptide to the fluorescent protein. The linker may be any moiety that provides for linking of the sensor polypeptide to the fluorescent protein moiety such as for example, a nucleic acid that encodes GGTGEL (SEQ ID NO:1) or FKTRHN (SEQ ID NO:2). Two or more linker moieties may be attached to two separate polypeptides, that together form a sensor polypeptide. Additionally, the indicator may have a localization sequence, for localizing the indicator, for example, to a particular organelle of a cell. The sensor polypeptide or linker moiety may be inserted at numerous sites including, for example, one or more amino acids between residues 128-148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein moiety (e.g., GFP). More particularly Y145 is used for insertion of the linker cr sensor polypeptide.
In another embodiment, the present invention provides a transgenic non-human animal having a nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.
In yet another embodiment, the present invention provides an expression vector having expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator. The indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.
In another embodiment, the present invention provides a host cell transfected with an expression vector having an expression control sequence operatively linked to a sequence coding for the expression of a fluorescent indicator. The host cell can be any host cell capable of transfection and expression of the indicator such as, for example, a prokaryote (e.g., E. coli), a eulcaryotic cell (e.g., a yeast cell) or a mammalian cell.
In yet a further embodiment, the present invention provides a fluorescent indicator polypeptide, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.
In another embodiment, the present invention provides a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide the responsiveness resulting in protonation or deprotonation of the chromophore of the fluorescent protein moiety.
In yet another embodiment, the present invention provides a method for detecting the presence of a environmental parameter in a sample, by contacting the sample with a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical, or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide, and detecting a change in fluorescence wherein a change is indicative of the presence of a parameter which affects the sensor polypeptide.
In another embodiment, the invention provides an isolated nucleic acid sequence encoding a circularly permuted fluorescent protein and the polypeptide encoded thereby, having a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
In another embodiment, the invention provides an expression vector comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator, the indicator having a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety, and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein. In a further embodiment, the invention provides a host cell containing the expression vector.
In yet another embodiment, the invention provides a method of producing a nucleic acid sequence encoding a fluorescent indicator, by linking a nucleic acid sequence encoding a linker moiety to the 5xe2x80x2 nucleotide of a polynucleotide encoding a fluorescent protein, circularizing the polynucleotide with the nucleic acid sequence encoding the linker sequence, and cleaving the circularized polynucleotide with a nuclease, wherein cleavage linearizes the circularized polynucleotide.
In yet another embodiment, the invention provides a method of producing a circularly permuted fluorescent protein by expressing a nucleic acid sequence encoding a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.